title: All-trans-retinoid acid (ATRA) may have inhibited chondrogenesis of primary hind limb bud mesenchymal cells by downregulating Pitx1 expression.
abstract: Despite frequently well-established role of all-trans-retinoid acid (ATRA) in congenital limb deformities, its mechanism of action, thus far, is still ambiguous. Pitx1, which is expressed in the hindlimb bud mesenchyme, or its pathways may be etiologically responsible for the increased incidence of clubfoot. Here, we sought to investigate the mechanisms whereby Pitx1 regulated chondrogenesis of hindlimb bud mesenchymal cells in vitro. E12.5 embryonic rat hind limb bud mesenchymal cells were treated with ATRA at appropriate concentrations. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Hematoxylin-safranin-O-fast-green staining assays were used to observe cartilage nodules, and Pitx1 expression was examined by immunofluorescent microscopy. Real-time quantitative PCR and immunoblotting assays were applied to determine the mRNA expressions of Pitx1, Sox9 and type II collagen (Col2al), respectively. The results showed that ATRA inhibited the proliferation of hind limb bud cells dose-dependently. ATRA also induced a dose-dependent reduction in the number of cartilage nodules and the area of cartilage nodules compared with controls. Our real-time quantitative RT-PCR assays revealed that the mRNA expression of Pitx1, Sox9 and Col2al were significantly downregulated by ATRA. Furthermore, our immunofluorescent microscopy and Western blotting assays indicated that Pitx1 was mainly expressed in the cartilage nodules and the levels of Pitx1, Sox9 and Col2al were also downregulated by ATRA dose-dependently. The results indicated that ATRA may decrease chondrogenesis of hind limb bud mesenchymal cells by inhibiting cartilage-specific molecules, such as Sox9 and Col2al, via downregulating Pitx1 expression.
authors.affiliation: Department of Orthopedics, The Second Hospital of Tianjin Medical University, Tianjin 300211, China.
authors.forename: Yun-guo
authors.initials: YG
authors.lastname: Wang
authors.forename: Xue-dong
authors.initials: XD
authors.lastname: Li
authors.forename: Zhao-yong
authors.initials: ZY
authors.lastname: Liu
authors.forename: Tao-gen
authors.initials: TG
authors.lastname: Zhang
authors.forename: Bin
authors.initials: B
authors.lastname: Chen
authors.forename: Guo-qing
authors.initials: GQ
authors.lastname: Hou
authors.forename: Quan
authors.initials: Q
authors.lastname: Hong
authors.forename: Peng
authors.initials: P
authors.lastname: Xie
authors.forename: Shi-xin
authors.initials: SX
authors.lastname: Du
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D020219:Chondrogenesis / Q000187:drug effects*; D024043:Collagen Type II / Q000037:antagonists & inhibitors / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005260:Female; D006614:Hindlimb / Q000196:embryology; D018878:Limb Buds / Q000166:cytology; D051761:Paired Box Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D055758:SOX9 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: C511513:COL2A1 protein, rat; D024043:Collagen Type II; D051761:Paired Box Transcription Factors; D055758:SOX9 Transcription Factor; C101485:homeobox protein PITX1; D014212:Tretinoin
keywords: Chondrogenesis; Clubfoot; Hind limb bud mesenchymal cells; Pitx1; Retinoic acid